WO2017223260A1 - Inhibiteurs de pu.1 - Google Patents

Inhibiteurs de pu.1 Download PDF

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WO2017223260A1
WO2017223260A1 PCT/US2017/038647 US2017038647W WO2017223260A1 WO 2017223260 A1 WO2017223260 A1 WO 2017223260A1 US 2017038647 W US2017038647 W US 2017038647W WO 2017223260 A1 WO2017223260 A1 WO 2017223260A1
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compound according
independently selected
cells
cycloalkyl
heterocyclyl
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PCT/US2017/038647
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W. David Wilson
David W. Boykin
Gregory POON
Ulrich Steidl
Iléana ANTHONY-DEBRÉ
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Albert Einstein College Of Medicine, Inc.
Georgia State University Research Foundation, Inc.
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Publication of WO2017223260A1 publication Critical patent/WO2017223260A1/fr
Priority to US16/227,739 priority Critical patent/US10774049B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/20—Two benzimidazolyl-2 radicals linked together directly or via a hydrocarbon or substituted hydrocarbon radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/12—Radicals substituted by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/18—Benzimidazoles; Hydrogenated benzimidazoles with aryl radicals directly attached in position 2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D421/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having selenium, tellurium, or halogen atoms as ring hetero atoms
    • C07D421/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having selenium, tellurium, or halogen atoms as ring hetero atoms containing three or more hetero rings

Definitions

  • This invention relates to novel inhibitors of myeloid master regulator PU.1.
  • ETS wenty-Six transcription factors
  • PU. l which regulates the expression of receptors such as IL-2Ry, IL-7Ra and Toll-like receptors.
  • PU. l is also involved in various autoimmune diseases. Because sequence specific binding is a necessary step in ETS-mediated gene activation, inhibition of the ETS-DNA complex can serve as the pharmacological basis for the treatment of a wide variety of diseases.
  • AML Acute myeloid leukemia
  • AML is a cancer of the hematopoietic system, characterized by the abnormal clonal proliferation of immature cells, following various genetic and epigenetic alterations.
  • survival in AML remains poor, with a 5-year overall survival of 25%, with overall outcome being worst for patients > 60 years of age who represent the vast majority.
  • clinical outcome has not significantly improved in the past 4 decades.
  • AML is a genetically very heterogeneous disease, characterized by recurrent genetic mutations which often occur in combination in individual patients (about 30 mutations recur in patients at a frequency of >1%), and on average patients with AML carry a combination of 3-5 'driver mutations'.
  • PU. l an ETS family transcription factor
  • a functionally critical decrease in PU. l level has been described in FLT3-ITD, RUNX1-ETO and promyelocytic leukemia, representing 24, 7 and 13% of all AMLs, respectively (cancer.sanger.ac.uk).
  • PU.1 loss of function heterozygous mutations or deletions have been described in AML, and are found in -10% of MLL-translocated AML. Homozygosity of a single nucleotide variant in an upstream regulatory element (URE) of PU. l , lowering PU.
  • URE upstream regulatory element
  • PU. l is highly conserved between humans and mice and its functions have been studied using a number of genetically engineered mouse models, which have further proven PU. l 's crucial role in hematopoiesis.
  • PU. l is essential for myeloid and lymphoid lineages, as well as hematopoietic stem cell (HSC) maintenance. Its role in AML development has been firmly established through mouse models with reduced, but not completely absent, PU. l expression.
  • Homozygous knockout of an enhancer (URE) located -14kb upstream of PU. l leads to a decrease in PU.1 expression of 80% and development of a stem cell-derived AML between 3 to 8 months of age.
  • Enhancer haplodeficiency of PU.1 is not sufficient to induce leukemia by itself; however it leads to myeloid bias in stem cells and AML development in combination with cooperating events.
  • PU. l and its downstream transcriptional network are crucial in hematopoiesis and leukemogenesis.
  • AML with disruption of PU. l function is a distinct entity, associated with specific oncogenes, as well as specific molecular signatures.
  • targeting PU.1 in AML could be an appealing option for treatment.
  • strategies to rescue PU.1 expression in AML cells have been explored.
  • Overexpression of PU.1 is sufficient to trigger neutrophil differentiation in acute promyelocytic leukemia (APL), and leads to differentiation and apoptosis of various primary AML samples.
  • APL acute promyelocytic leukemia
  • elevation of PU. l levels or activity is difficult to achieve pharmacologically.
  • AML cells may be more vulnerable to further PU. l inhibition in comparison to normal hematopoietic cells.
  • PU.1 is also a promising target in a range of non-malignant diseases with an immunological basis, in which pharmacological inhibition represents a novel therapeutic strategy.
  • the essential role of PU. l in the differentiation and development of myeloid lineages is well established in mouse and human models of hematopoiesis.
  • PU.1 induces the expression of key receptors such as TLR4 and GM-CSFR, which sensitize granulocytes and monocytes to endotoxins and specific pro-inflammatory cytokines.
  • TLR4 and GM-CSFR key receptors
  • PU. l represents an attractive therapeutic target in non-malignant inflammatory diseases in which granulocytes and monocytes are major cellular mediators. Examples of such diseases that are mediated, at least in part, by granulocytes/monocytes include (but are not restricted to) endotoxemia, rheumatoid arthritis and neurodegenerative diseases.
  • GM-CSF stimulates differentiation of tissue macrophages and sensitization to bacterial endotoxins (LPS) in a PU.
  • LPS bacterial endotoxins
  • endotoxins potently stimulates TLR4 on mature macrophages, leading to local (e.g., lung) and systemic inflammation that is blunted in PU.
  • l -deficient chimeric animals In other mouse models, endotoxins potently stimulates TLR4 on mature macrophages, leading to local (e.g., lung) and systemic inflammation that is blunted in PU. l -deficient chimeric animals.
  • IL- 9 Secretion of IL- 9 by Th9 cells, a major cytokine of in allergic inflammation, is transcriptionally controlled by PU.1 following induction by TGF .
  • Evidence is rapidly accumulating that IL-9 is the mediator in acute contact dermatitis, asthma, inflammatory bowel disease, pediatric atopy, and giant cell arteritis.
  • PU.1 In addition to ensuring the self-renewal of the hematopoietic stem cell (HSC), PU.1 governs cell fate determination in a dosage- and cell-stage dependent fashion. Elevated PU.1 activity is required to drive differentiation of the HSC towards the myeloid lineages (the common myeloid progenitor), at which point continued PU. l activity induces the terminal development of macrophages and granulocytes, while a tapering of PU.1 activity leads to erythrocytes. At lower concentrations, PU. l also drives the initial differentiation of the HSC to the common lymphoid progenitor, at which stage a switch in PU.1 dosage induces terminal differentiation into B- (high PU.
  • HSC hematopoietic stem cell
  • PU. l T-lymphocytes
  • PU. l inhibitors are expected to be useful in a cocktail of other transcriptional modulators, to induce the differentiation of appropriate progenitors into desired cell types.
  • the expected usefulness of PU. l inhibitors as a cell-reprogramming agent is highlighted by a reversal in PU. l/Ets-1 antagonism in the specialization of mature T cells into subtypes such as Th9.
  • DNA site recognition by PU. l requires contact with the major groove, at consensus sites harboring the 5'-GGAA/T-3' sequence specific for the ETS family. Additional contacts with the adjacent DNA minor groove confer selectivity for certain ETS paralogs, such as AT-rich sequences for PU. l. PU. l inhibitors targeting DNA in the minor groove, by targeting the AT-rich sequences, therefore lead to inhibition of PU.1 binding in the major groove via an allosteric mechanism.
  • PU.1 inhibitors capable of inhibiting PU. l.
  • the PU.1 inhibitors can be characterized by the following chemical formula:
  • x and x' are each 3;
  • R is in each case independently selected from R a , OR a , N(R a ) 2 , SR a , S0 2 R a , S0 2 N(R a ) 2 ;
  • R a is in each case independently selected from hydrogen, C 1-8 alkyl, C 3 -8 cycloalkyl, C 2- g heterocyclyl, C 6 -i 2 aryl, and C 3-12 heteroaryl, wherein any two or more of R and R a may together form a ring;
  • G and G' are independently selected from C3-8 cycloalkyl, C 2- g heterocyclyl, C 6 -i 2 aryl, and C 3-12 heteroaryl;
  • a and A' are independently selected from NR 1 , O, S, and Se, wherein R 1 , when present, is in each case independently selected from R b , S0 2 R b , S0 2 N(R b ) 2 ; COOR b , C(0)N(R b ) 2 , wherein R b is in each case independently selected from hydrogen, C 1-8 alkyl, C 3 -8 cycloalkyl, C 2- g heterocyclyl, C 6 -i 2 aryl, and C 3-12 heteroaryl, wherein any two or more of R and R 1 may together form a ring;
  • B and B' are independently selected from N and CR;
  • Q a is O or NR a , wherein R a , R a , and R a are independently selected from hydrogen, Ci-8 alkyl, C3-8 cycloalkyl, C 2- 8 heterocyclyl, C 6- i 2 aryl, and C 3-12 heteroaryl; wherein any two or more of R la , R 2a , R a , R and R 1 can together form a ring;
  • Q b is O or NR lb , wherein R lb , R 2b , and R b are independently selected from
  • Z is a linking group having the formula:
  • X and Y are independently selected from: a chemical bond; O, S, Se, and NR 4 ; wherein R 4 , when present, is in each case independently selected from R c , SC ⁇ R 0 , S0 2 N(R c ) 2 ; COOR c , C(0)N(R c ) 2 , wherein R c is in each case independently selected from hydrogen, C 1-8 alkyl, C3-8 cycloalkyl, C2-8 heterocyclyl, Ce-u aryl, and C 3-12 heteroaryl;
  • R2 is in each case independently H or F or mixtures thereof;
  • n are each an integer independently selected from 0-4;
  • C z is selected from a chemical bond, O, S, Se, NR 4 , or C2-8 heterocyclyl, C6-12 aryl, and C 3-12 heteroaryl;
  • the compounds disclosed herein are PU.1 inhibitors, and as such can be used to treat diseases associated with abnormal PU. l activity.
  • Fig. 1A-1F. PU. l knockdown decreases cell growth and increases apoptosis of murine and human PU. l low leukemic cells.
  • (C) Apoptotic cells (Annexin-V+ Dapi-) fraction in URE-/- cells after transduction with shPU. l l and shPU. l_2 (n 3). Data represent the means ⁇ SD of independent experiments; fold change compared to shCtrl is shown.
  • Fig. 2A-2F. PU. l inhibition decreases cell growth and increases apoptosis of PU. l low leukemic cells.
  • A Chemical structures of heterocyclic diamidines DB1976, DB21 15 and DB2313 used in this study.
  • B Relative cell viability of URE-/- cells and BAF3 cells after treatment with increasing concentrations of vehicle or small molecules. Cell viability was assessed after 48h. Data represent means of technical triplicate from one representative experiment; fold change compared to Vehicle is shown.
  • FIG. 3A-3G Novel inhibitors show on-target PU. l inhibitory activity.
  • B Chromatin immunoprecipitation assay showing PU.
  • E,F Comparison analysis of deregulated genes in URE-/- cells after DB2313 treatment and in PUER cells after PU. l induction (GSE13125). Deregulated genes in PUER cells were analyzed with a FC at 1.5 and P-value ⁇ 0.05.
  • E Significant overlap between the 2 datasets, with 484 genes commonly deregulated. P-value was calculated using the hypergeometric test.
  • F Comparative analysis of deregulated canonical pathways between the 2 datasets (z-score > 2 for PUER dataset).
  • Fig. 4A-4G. PU. l inhibition decreases cell growth and increases apoptosis of primary human AML cells.
  • A-C Transduced GFP+ human primary mononuclear AML cells were plated in semi-solid media (150 000 cells per ml of methyl); colony number, number of viable cells and apoptotic cells were assessed after 14 days of culture.
  • D-F Human primary mononuclear AML cells were plated in semi-solid media (150 000 cells per ml of methyl) containing the PU. l inhibitors DB1976, DB2115 and DB2313; colony number, number of viable cells and apoptotic cells were assessed after 14 days of culture.
  • G Enrichment of PU. l binding in up- and down-regulated genes.
  • A-G Data represent means ⁇ SD, each AML sample is represented by an individual dot. Fold change compared to shCtrl or Vehicle is shown.
  • Fig. 5A-5F. PU. l inhibitors decrease the granulo-monocytic potential of hematopoietic stem and progenitor cells, which is rapidly reversible.
  • A-C Lin-Scal+c-Kit+ (LSK) cells were plated in semi-solid media containing PU. l inhibitors.
  • A Number of colony-forming unit granulocyte (CFU-G), monocyte (CFU-M), granulo/monocyte (CFU- GM), granulo/erythrocyte/monocyte/megakaryocyte (CFU-GEMM), burst or colony forming unit erythroid (B/CFU-E) and immature colonies after treatment.
  • CFU-GM, CFU-G and CFU-M numbers are shown. Data represent the means ⁇ SD of 3 independent experiments.
  • B Cells coming from colony assays after vehicle or DB2313 treatment. Cells were cytospun and stained with May-Griinwald Giemsa. Scale bar is equal to 20 ⁇ .
  • C FACS analysis showing the percentage of CDl lb+Grl-, CDl lb+Grl+, CDl lb- Grl+, CD41+Terl l9-, CD41+Terl l9+, CD41-Terl l9+ populations after colony assays. Data represent the means ⁇ SD of 4 independent experiments.
  • FIG. 6A-6H Treatment with PU1 inhibitors increases survival and decreases tumor burden in vivo.
  • A Experimental scheme. URE-/- cells were treated in vitro with DB2313 or Vehicle and counted after 2 days of culture. 200.10 3 viable cells per mouse were injected retroorbitally into sublethally irradiated mice. Mice were sacrificed at 6 weeks or used for survival analyses.
  • C,D Spleen and liver weight 6 weeks after transplant (8 animals for vehicle group, 7 for DB2313 group).
  • Fig. 7 depicts the effects of PU.1 inhibition of AML and healthy hematopoietic cells.
  • alkyl as used herein is a branched or unbranched hydrocarbon group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, and the like.
  • the alkyl group can also be substituted or unsubstituted. Unless stated otherwise, the term "alkyl” contemplates both substituted and unsubstituted alkyl groups.
  • the alkyl group can be substituted with one or more groups including, but not limited to, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, or thiol as described herein.
  • An alkyl group which contains no double or triple carbon-carbon bonds is designated a saturated alkyl group, whereas an alkyl group having one or more such bonds is designated an unsaturated alkyl group.
  • Unsaturated alkyl groups having a double bond can be designated alkenyl groups, and unsaturated alkyl groups having a triple bond can be designated alkynyl groups. Unless specified to the contrary, the term alkyl embraces both saturated and unsaturated groups.
  • cycloalkyl as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms.
  • examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.
  • heterocycloalkyl is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, selenium or phosphorus.
  • the cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted.
  • cycloalkyl and heterocycloalkyl contemplate both substituted and unsubstituted cycloalkyl and heterocycloalkyl groups.
  • the cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, or thiol as described herein.
  • a cycloalkyl group which contains no double or triple carbon-carbon bonds is designated a saturated cycloalkyl group, whereas an cycloalkyl group having one or more such bonds (yet is still not aromatic) is designated an unsaturated cycloalkyl group.
  • the terms cycloalkyl and heterocycloalkyl embrace both saturated and partially unsaturated systems.
  • aryl as used herein is an aromatic ring composed of carbon atoms. Examples of aryl groups include, but are not limited to, phenyl and naphthyl, etc.
  • heteroaryl is an aryl group as defined above where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, selenium or phosphorus.
  • the aryl group and heteroaryl group can be substituted or unsubstituted. Unless stated otherwise, the terms “aryl” and “heteroaryl” contemplate both substituted and unsubstituted aryl and heteroaryl groups.
  • the aryl group and heteroaryl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, or thiol as described herein.
  • heteroaryl and heterocyclyl rings include: benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cirrnolinyl, decahydroquinolinyl, 2H,6H ⁇ 1 ,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, lH-indazolyl, indolenyl, indolinyl,
  • alkoxy has the aforementioned meanings for alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, further providing said group is connected via an oxygen atom.
  • the term "substituted" is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms, such as nitrogen can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds.
  • substitution or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g. , a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • a substituent that is said to be “substituted” is meant that the substituent is substituted with one or more of the following: alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, or thiol as described herein.
  • the term "patient” refers to any mammalian animal, including but not limited to, humans.
  • salts are salts that retain the desired biological activity of the parent compound and do not impart undesirable toxicological effects.
  • examples of such salts are acid addition salts formed with inorganic acids, for example, hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric acids and the like; salts formed with organic acids such as acetic, oxalic, tartaric, succinic, maleic, fumaric, gluconic, citric, malic, methanesulfonic, p-toluenesulfonic, napthalenesulfonic, and polygalacturonic acids, and the like; salts formed from elemental anions such as chloride, bromide, and iodide; salts formed from metal hydroxides, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, lithium hydroxide, and magnesium hydroxide; salts formed from metal carbonates, for example, sodium carbonate, potassium carbonate, calcium carbonate, and magnesium carbonate; salts
  • Pharmaceutically acceptable and non-pharmaceutically acceptable salts may be prepared using procedures well known in the art, for example, by reacting a sufficiently basic compound such as an amine with a suitable acid comprising a physiologically acceptable anion.
  • a sufficiently basic compound such as an amine
  • a suitable acid comprising a physiologically acceptable anion.
  • Alkali metal for example, sodium, potassium, or lithium
  • alkaline earth metal for example, calcium
  • Human Transcription factor PU. l protein (product of the SPI1 gene is) has the following amino acid sequence (SEQ ID NO: l) (UniProtKB - P17947):
  • x and x' are each 3;
  • R is in each case independently selected from R a , OR a , N(R a ) 2 , SR a , S0 2 R a , S0 2 N(R a ) 2 ;
  • R a is in each case independently selected from hydrogen, C 1-8 alkyl, C3-8 cycloalkyl, C 2- g heterocyclyl, C6-i 2 aryl, and C 3-12 heteroaryl, wherein any two or more of R and R a may together form a ring;
  • G and G' are independently selected from C3-8 cycloalkyl, C 2- g heterocyclyl, C6-i 2 aryl, and C 3-12 heteroaryl;
  • a and A' are independently selected from NR 1 , O, S, and Se, wherein R 1 , when present, is in each case independently selected from R b , S0 2 R b , S0 2 N(R b ) 2 ; COOR b , C(0)N(R b ) 2 , wherein R b is in each case independently selected from hydrogen, C 1-8 alkyl, C3-8 cycloalkyl, C 2- g heterocyclyl, C6-i 2 aryl, and C 3-12 heteroaryl, wherein any two or more of R and R 1 may together form a ring
  • B and B' are independently selected fromN and CR;
  • Q a is O or NR la , wherein R la , R 2a , and R a are independently selected from hydrogen, Ci-8 alkyl, C3-8 cycloalkyl, or C2-8 heterocyclyl; wherein any two or more of R la , R 2a , R a , R and R 1 can together form a ring;
  • Q b is O or NR lb , wherein R lb , R 2b , and R b are independently selected from hydrogen, C 1-8 alkyl, C3-8 cycloalkyl, and C2-8 heterocyclyl; wherein any two or more of R lb , R 2b , and R b , R and R 1 can together form a ring;
  • Z is a linking group having the formula:
  • X and Y are independently selected from: a chemical bond; O, S, Se, and NR 4 ; wherein R 4 , when present, is in each case independently selected from R c , SC ⁇ R 0 , S0 2 N(R c ) 2 ; COOR c , C(0)N(R c ) 2 , wherein R c is in each case independently selected from hydrogen, C 1-8 alkyl, C3-8 cycloalkyl, C2-8 heterocyclyl, Ce-u aryl, and C 3-12 heteroaryl
  • R2 is in each case independently H or F or mixtures thereof;
  • n are each an integer independently selected from 0-4;
  • C z is selected from a chemical bond, O, S, Se, NR 4 , C3-8 cycloalkyl, C2-8 heterocyclyl, C6-12 aryl, and C 3-12 heteroaryl;
  • one or both of X and Y are O.
  • B and B' are both N.
  • a and A' are both NR 4 , in which R 4 is either hydrogen or C 1-4 alkyl.
  • both of X and Y are O, B and B' are both N, and A and A' are both NR 4 , in which R 4 is either hydrogen or C 1-4 alkyl.
  • one or both of the a and ⁇ groups can be in the 4 position
  • one or both of the a and ⁇ groups can be in the 5, 6 or 7 position.
  • the phenyl portion of the bicyclic heterocycle portion of the compound may be substituted only with a and ⁇ , that is, each of the R groups in those rings is hydrogen, for instance a compound having the formula:
  • At least one R group is not hydrogen.
  • one of the R groups can be an electron donating group in ortho, meta or para position relative to the a or ⁇ substituent.
  • Exemplary electron donating groups include R a , OR a , N(R a ) 2 .
  • one of the R groups can be an electron withdrawing group in the ortho, meta or para relative to the a or ⁇ substituent.
  • Exemplary electron withdrawing groups include S0 2 R a , S0 2 N(R a ) 2 ; COOR a , C(0)N(R a ) 2 , OC(0)N(R a ) 2 , N(R a )C(0)N(R a ) 2 , F, CI, Br, I, cyano, and nitro.
  • G and G' can be an optionally substituted phenyl group or an optionally substituted heteroaryl.
  • G and G' can be independently selected from:
  • R 6 is independently selected from R d , OR d , N(R d ) 2 , SR d , S0 2 R d , S0 2 N(R d ) 2 ; COOR d , C(0)N(R d ) 2 , OC(0)N(R d ) 2 , N(R d )C(0)N(R d ) 2 , F, CI, Br, I, cyano, and nitro, wherein R d is in each case independently selected from hydrogen, C 1-8 alkyl, C3-8 cycloalkyl, C 2- g heterocyclyl, C6-i 2 aryl, and C 3-12 heteroaryl, wherein any two or more of R and R a may together form a ring.
  • R 6 is in each case hydrogen.
  • G or G' can be a phenyl or heteroaryl having one or two non-hydrogen groups.
  • the non-hydrogen R 6 group can be selected from R d , OR d , COOR d , F, CI, Br, I, cyano, and nitro.
  • the four G and G' systems described above can be designated 1,4 systems by virtue that the connectivity pattem in the para configuration.
  • G and G' can be optionally substituted phenyl, pyridinyl or 1,3 pyrazine group in the 1,3 or 1,2 configuration.
  • the substitution pattem can also be in the 1,2 configuration.
  • a and ⁇ are both at the 6 position, and in further embodiments can have an electron withdrawing group at the 4, 5 or 7 position. In some embodiments, a and ⁇ are both at the 6 position, and in further embodiments have an electron donating group at the 4, 5, or 7 position.
  • Z has the formula:
  • Z can be O-CH2-CF2-CF2-CF2-CH2-O-; 0-CH 2 -CF 2 -CF 2 -0-; -0-CF 2 -CF 2 -CF 2 -0-;
  • C z can be an optionally substituted phenyl or heteroaryl.
  • C z can have the formula: , wherein
  • A" is O, S, Se, or NR 6 ; wherein R 6 is hydrogen, Ci-galkyl; C3-8 cycloalkyl, and C2-8 heterocyclyl; and in other instances C z can have the formula:
  • C z can be an optionally substituted phenyl or heteroaryl.
  • C z can have the formula: ( r7 )2 wherein R 7 is independently selected from hydrogen, F, CI, Br, I, cyano or nitro.
  • C z can have the formula:
  • R has the meaning given above.
  • R can be R 7 .
  • R can be R 7 , in which R 7 is in each case hydrogen.
  • C z can be a phenyl group having the formul
  • R has the meaning given above.
  • R can be R 7 .
  • R can be R 7 , in which R 7 is in each case hydrogen.
  • C z can be a phenyl group having the formula:
  • R 7 can be R, or can be selected from hydrogen, F, CI, Br, I, cyano or nitro, preferably F.
  • C z can be selected from a chemical bond, O, S, Se, NR 4 , C3-8 cycloalkyl, C2-8 heterocyclyl, C 6-12 aryl, and C 3-12 heteroaryl or a group of the formula:
  • A" is O, S, Se, or NR ; wherein R is hydrogen, Ci-salkyl; C3-8 cycloalkyl, and C2-8 heterocyclyl, wherein when X and Y are both O, C z is not a chemical bond, and when A" is Se, X and Y are not both a chemical bond.
  • a has the formula:
  • ⁇ has the formula:
  • the compounds disclosed herein may be formulated in a wide variety of compositions for administration to a patient, for instance, a human patient.
  • the compounds disclosed herein are especially useful in treating disease states in elderly patients, i.e., those of sixty years of age or greater.
  • the compounds can be delivered, for example, orally, intravenously, topically, parentally, subcutaneously, intradermally, or by inhalation.
  • Exemplary routes of administration include buccal, oral, intravenous, intramuscular, topical, subcutaneous, rectal, vaginal, parenteral, pulmonary, intranasal, ophthalmic, and the like.
  • compositions comprising any of the compounds disclosed herein and a pharmaceutically acceptable carrier.
  • carrier is used in accordance with its art-understood meaning, to refer to a material that is included in a pharmaceutical composition but does not abrogate the biological activity of pharmaceutically active agent(s) that are also included within the composition.
  • carriers typically have very low toxicity to the animal to which such compositions are to be administered. In some embodiments, carriers are inert.
  • Pharmaceutically acceptable carriers and diluents that can be used herewith encompasses any of the standard pharmaceutical carriers or diluents, such as, for example, a sterile isotonic saline, phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsions.
  • a medicament comprising any of the compounds disclosed herein or any of the pharmaceutical compositions disclosed herein, wherein the compound is in an amount effective to inhibit PU.1.
  • the compounds and compositions disclosed herein may be used to treat diseases associated with abnormal PU. l levels and activity.
  • the compounds can be used to treat consisting of hematologic cancer, bone cancer, inflammatory disease, inflammatory disorders, autoimmune disorders, endotoxemia and neurodegenerative disease. Exemplary such conditions include acute myeloid leukemia, rheumatoid arthritis, contact dermatitis, asthma, inflammatory bowel disease, pediatric atrophy, giant cell arteritis, Alzheimer's disease, and systemic lupus.
  • the invention provides a method of treating a patient with a disease associated with abnormal PU. l function, comprising administering to the patient in need thereof any of the compounds or compositions disclosed herein in an amount effective to inhibit PU. l .
  • the patient can be a human patient or a veterinary patient.
  • the human patient can be, for example, at least 60 years old.
  • "treat" a disease means to ameliorate a sign or symptom of the disease, or to cure the patient of the disease.
  • Useful dosages of the compounds of the invention for inclusion in the pharmaceutical compositions of the invention can be determined by comparing in vitro activity and in vivo activity of the compounds in appropriate animal models.
  • concentration of the compound(s) of the invention in a liquid composition will range from about 0.1 % to about 95 % by weight, preferably from about 0.5 % to about 25 % by weight.
  • concentration in a semi- solid or solid composition will range from about 0.1 % to 100% by weight, preferably about 0.5 % to about 5 % by weight.
  • Single doses for intravenous injection, subcutaneous, intramuscular or topical administration, infusion, ingestion or suppository will generally be from about 0.001 to about 5000 mg, and be administered from about 1 to about 3 times daily, to yield levels of about 0.01 to about 500 mg/kg, for adults.
  • the compounds can be co-administered with one or more other agents for the treatment of any of the aforementioned diseases.
  • the one or more other agents is a transcription modulator.
  • the one or more other agents can be immunosuppressants.
  • the other agents can be formulated separately, and administered either at the same or different time as the compounds of the instant invention.
  • the other agents can be co-formulated with the compounds of the instant invention to give a combination dosage form.
  • DB2115 and DB2313 were dissolved as a 2.5 mM, 70uM and 33 (mouse cells) or 66uM (human) uM stock solutions, respectively, in sterile water and stored at -20°C.
  • Example 2 PU. l knockdown decreases cell growth and clonogenic capacity, and increases apoptosis of murine and human AML cells.
  • PU. l inhibition may be a suitable strategy in AML
  • URE upstream enhancer
  • Knockdown of PU. l in PU. l URE _ " AML cells by two different shRNAs led to significantly decreased cell growth and colony formation (Fig. 1A, IB).
  • the percentage of apoptotic cells was significantly increased upon shRNA- mediated PU. l knockdown in PU. l URE- ⁇ - AML cells (Fig. 1C).
  • the K ⁇ ) values indicate strong binding by all three compounds to form a 1 : 1 complex, and a binding K ⁇ ) of 1 ⁇ 2 nM for DB2115 and 4-7 nM for DB1976 and DB2313 with the ⁇ promoter.
  • a binding K ⁇ 1 ⁇ 2 nM for DB2115 and 4-7 nM for DB1976 and DB2313 with the ⁇ promoter.
  • DB2313 binds to the AT sequence that is on the 5' side of the central 5' -GGAA-3' conserved PU. l recognition site.
  • PU. l has a "winged helix" DNA recognition motif with the helix in the major groove at the GGAA and wings that contact the 5' AT sequence as well as the 3' side of GGAA.
  • the model shows DB2313 inserts deeply into the minor groove in a way that locks the DNA structure into a specific conformation.
  • DB2313 has the ability to recognize 10 consecutive base pairs in the minor groove and interacts strongly with the DNA base pair edges at the floor of the minor groove. This interaction interferes with the PU. l complex in the major groove and causes dissociation of PU. l in an allosteric process.
  • l inhibitors led to a significant 1.6-, 2- and 3.5-fold increase of apoptotic cells with DB1976, DB2115 and DB2313, respectively, in murine PU.
  • l URE _ AML cells (Fig. 3C), and similar effects in human MOLM13 cells.
  • PU. l inhibitors also significantly decreased colony forming capacity of PU. l
  • PU. l inhibitors led to significant decreases in the number of viable cells (mean decrease: 81% for DB1976, 68% for DB2115, 72% for DB2313) (Fig. 3E), and clonogenic capacity (mean decrease of 36% for DB1976, 45% for DB21 15, 60% for DB23313), in comparison to vehicle (Fig. 3F).
  • the apoptotic cell fraction increased on average by 1.5-fold with DB 1976, 2.2-fold with DB21 15 and 2.5-fold with DB2313 (Fig. 3G).
  • PU. l inhibition had an effect on the maj ority of samples, harboring various genetic and cytogenetic anomalies.
  • l targets in myeloid cells we assessed its expression at different time points (lh, 4h, 8h and 24h) after drug treatment.
  • Csflr expression significantly decreased as early as 4h after treatment (Fig. 4A, right panel), in line with a direct effect of the drugs on PU. l transcriptional activity.
  • treatment of bone marrow mononuclear cells isolated from a PU. l -GFP knock-in reporter mouse model led to a decrease in GFP reporter expression after treatment with DB1976, DB21 15, or DB2313, further confirming a direct effect of the compounds on PU.1 transactivation (Fig. 4B), and consistent with PU.1 positive autoregulation.
  • Chromatin-immunoprecipitation (ChIP) assays revealed that treatment of AML cells with DB1976, DB2115 or DB2313 indeed decreased PU. l occupancy on E2fl, Junb and Csflr promoters, confirming that the compounds are directly interfering with PU.1 binding to chromatin in vivo (Fig. 4C).
  • l target genes such as Ly96, Clec5a, Cdknl a, Itgb2, Fcgr3 and Gfil , by qRT-PCR.
  • cytomorphologic analysis revealed almost no macrophages, significantly fewer mature granulocytes, and an increase in immature cells, characterized by a higher nucleocytoplasmic ratio and basophilic cytoplasm after treatment (Fig. 5B). This was confirmed by flow cytometric analysis, which revealed a reduction in mature monocytes
  • Example 7 Treatment with PU.1 inhibitors decreases leukemia progression in vivo
  • FIG. 6A AML cells for 2 days in vitro and injected 2x10 ⁇ viable cells in sublethally irradiated recipient mice
  • Fig. 6B Assessment of tumor burden 6 weeks post transplantation showed a decrease in spleen and liver weights after treatment; splenic mean weight was 410 mg for vehicle and 243 mg for DB2313 (Fig. 6C); liver mean weight was 2,347 mg for vehicle and 1,548 mg for DB2313 (Fig. 6D).
  • AML cell engraftment in the bone marrow was significantly decreased after treatment with DB2313, with a mean chimerism of 55% for the vehicle group and 33% for DB2313 group (Fig. 6E, 6F). Histological analyses revealed severe blast infiltration with disruption of the splenic architecture and virtually complete loss of the red pulp in the vehicle group, and significantly less pronounced effects in the DB2313 group (Fig. 6G). Likewise, infiltration of the liver by leukemic blasts was substantially reduced in the DB2313 group in comparison to the vehicle group (Fig. 6H).
  • compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims.
  • Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims.

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Abstract

L'invention concerne des inhibiteurs de PU.1 qui sont utiles pour traiter des troubles associés à des niveaux et à une fonction de PU.1 anormaux.
PCT/US2017/038647 2016-06-23 2017-06-22 Inhibiteurs de pu.1 WO2017223260A1 (fr)

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WO2022105825A1 (fr) * 2020-11-20 2022-05-27 Peking University Composés en tant qu'inhibiteurs de pu.1
CN114588152A (zh) * 2022-04-14 2022-06-07 安徽医科大学 Pu.1抑制剂db2313的用途
US11827627B2 (en) 2021-06-04 2023-11-28 Vertex Pharmaceuticals Incorporated N-(hydroxyalkyl (hetero)aryl) tetrahydrofuran carboxamides as modulators of sodium channels
US11834441B2 (en) 2019-12-06 2023-12-05 Vertex Pharmaceuticals Incorporated Substituted tetrahydrofurans as modulators of sodium channels
EP4028028A4 (fr) * 2019-09-13 2023-12-27 Massachusetts Institute of Technology Systèmes et essais pour identifier des inhibiteurs de pu.1

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Cited By (6)

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Publication number Priority date Publication date Assignee Title
EP4028028A4 (fr) * 2019-09-13 2023-12-27 Massachusetts Institute of Technology Systèmes et essais pour identifier des inhibiteurs de pu.1
US11834441B2 (en) 2019-12-06 2023-12-05 Vertex Pharmaceuticals Incorporated Substituted tetrahydrofurans as modulators of sodium channels
US11919887B2 (en) 2019-12-06 2024-03-05 Vertex Pharmaceuticals Incorporated Substituted tetrahydrofurans as modulators of sodium channels
WO2022105825A1 (fr) * 2020-11-20 2022-05-27 Peking University Composés en tant qu'inhibiteurs de pu.1
US11827627B2 (en) 2021-06-04 2023-11-28 Vertex Pharmaceuticals Incorporated N-(hydroxyalkyl (hetero)aryl) tetrahydrofuran carboxamides as modulators of sodium channels
CN114588152A (zh) * 2022-04-14 2022-06-07 安徽医科大学 Pu.1抑制剂db2313的用途

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